An Active-Site Phenylalanine Directs Substrate Binding and C−H Cleavage in the α-Ketoglutarate-Dependent Dioxygenase TauD

• Published in: Journal of the American Chemical Society Vol. 132; no. 14; pp. 5114 - 5120
• Main Authors: McCusker, Kevin P, Klinman, Judith P
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 14.04.2010
• Subjects: Carbon - metabolism; Catalytic Domain; Ferrous Compounds; Hydrogen - metabolism; Kinetics; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - metabolism; Models, Molecular; Phenylalanine - metabolism; Substrate Specificity
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja909416z

Enzymes that cleave C−H bonds are often found to depend on well-packed hydrophobic cores that influence the distance between the hydrogen donor and acceptor. Residue F159 in taurine α-ketoglutarate dioxygenase (TauD) is demonstrated to play an important role in the binding and orientation of its substrate, which undergoes a hydrogen atom transfer to the active site Fe(IV)O. Mutation of F159 to smaller hydrophobic side chains (L, V, A) leads to substantially reduced rates for substrate binding and for C−H bond cleavage, as well as increased contribution of the chemical step to k cat under steady-state turnover conditions. The greater sensitivity of these substrate-dependent processes to mutation at position 159 than observed for the oxygen activation process supports a previous conclusion of modularity of function within the active site of TauD (McCusker, K. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 19791−19795). Extraction of intrinsic deuterium kinetic isotope effects (KIEs) using single turnover transients shows 2- to 4-fold increase in the size of the KIE for F159V in relation to wild-type and F159L. It appears that there is a break in behavior following removal of a single methylene from the side chain of F159L to generate F159V, whereby the protein active site loses its ability to restore the internuclear distance between substrate and Fe(IV)O that supports optimal hydrogenic wave function overlap.